Nucleosomes tightly wrap 75-90% of eukaryotic DNA, causing the DNA to be largely inaccessible to polymerases, regulatory and recombination complexes, thereby antagonizing gene expression. Experiments have demonstrated that DNA has sequence preference for positioning of nucleosomes, strongly suggesting that DNA has an inherent code that dictates where nucleosomes are most likely located. Nucleosomes and transcription factors are often in competition for binding the same regions of DNA and this competition creates a delicate balance between antagonizing and promoting gene expression, respectively. Recently, a number of nucleosome mapping projects have provided a plethora of information regarding genomic sequences that nucleosomes favor and disfavor and make many different kinds of concrete and powerful predications concerning how genomes use their intrinsically encoded nucleosome organization to regulate gene expression. The goal of this proposal is to elucidate key fundamental cellular mechanisms for controlling gene expression: (1) determine the role for nucleosome-rich TATA-box regions in antagonizing gene expression, (2) determine the role for nucleosome-depleted poly(dA:dT) tracts in promoting gene expression and (3) determine the role for nucleosome architecture in evolution. I will make mutations in promoter regions to alter the affinity of a nucleosome for that region of DNA and determine if the nucleosome occupancy changes as predicted based on the mutation. Further, I will analyze if this change in nucleosome occupancy alters transcription factor binding. Finally, I will monitor if changes in nucleosome occupancy do indeed effect gene expression. For aims one and two, I will use two model promoter systems, PH05 and GAL1,10, in the model organism S. cerevisiae. For aim 3, I will compare the role of the nucleosome code by swapping the genetic information from a promoter region of a respiratory gene of an anaerobic yeast species into an aerobic yeast species. These respiration genes have significantly different expression levels in the two yeast species. A greater understanding of the role of the competition between nucleosome and transcription factor binding of DNA on protein expression will provide significant insight into regulation of protein production, a necessary component for preventing cancer. Proteins are often considered the workhorses of the cell, and it is essential to regulate the production of proteins for the development and health of an organism. A lack of regulation can lead to uncontrolled cell growth, a trait of cancerous cells. This proposal focuses on understanding how protein expression is regulated by determining what controls the availability of the DNA for protein production.